JP2014223084A - Antibodies - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide antibodies and their derivatives that induce apoptosis upon binding to P-Selectin Glycoprotein Ligand-1 (PSGL-1) on activated T cells.SOLUTION: There are disclosed immunoglobulin chains or antibodies having light or heavy chain complementarity determining regions of antibodies that bind to P-Selectin Glycoprotein Ligand-1. Also disclosed are nucleic acids encoding the immunoglobulin chains, vectors and host cells having the nucleic acids, and methods of inducing death of an activated T cell and methods of modulating a T cell-mediated immune response in a patient.

Description

This application claims priority to US Provisional Patent Application No. 60 / 569,892, filed May 10, 2004, which is hereby fully incorporated by reference. .

Background Very aggressive T cells often cause undesirable immune responses, thereby causing various diseases such as autoimmune diseases, transplant rejection, allergic diseases, and T cell-induced cancers. Therefore, aggressive T cell control is important in treating such diseases. The activity of these cells can be blocked by immunosuppression or by induction of immune tolerance. Another solution is induction of apoptosis, which is believed to be involved in removing unwanted cells, such as highly aggressive T cells. For example, Kabelitz et al. (1993) Immunol Today 14, 338-340; and Raff (1992) Nature 356, 397-399.

SUMMARY The present invention relates to antibodies and derivatives thereof that induce apoptosis when bound to P-selectin glycoprotein ligand-1 (PSGL-1) of activated T cells.

  In one aspect, the invention includes (i) RSSQSIVHNDGNTYFE, KVSNRRFS, and FQGSHVVPLT (SEQ ID NOs: 1-3), respectively; (ii) SFGMH, YINGGSSTIFYANAVGKG, and YASYGGGAMDY (SEQ ID NOs: 4-6, respectively) (Iii) includes RASTVNSTYLH, GSSNLAS, and QQYSGYPLT (SEQ ID NOs: 7-9), respectively; (iv) includes AYYIH, VNPNTGTTSNPNPKFKG, and SGSPYYRYDD (SEQ ID NOs: 10-12), respectively; ) Each of which includes RSSQSIVNSNGNTYLE, KVSNRRFS, and FQDNSHVPWT (SEQ ID NO: 13-15); or (vi) TNMNWVR, respectively. APGKGLE, TYYADSVKD, and GGSYWYFDV (SEQ ID NO: 16-18) containing, characterized immunoglobulin chain having the sequence of the three.

  The six sets of sequences are complementary to the three light or heavy chains of an antibody that binds to PSGL-1, such as those of the three mouse 15A7, 43B6, and 9F9 antibodies described in the Examples below. It corresponds to a decision region (CDR). The light and heavy chain variable (V) regions of these three antibodies are shown below (SEQ ID NOs: 19-26, CDRs are underlined and highlighted):

  Since the antigen binding specificity of an antibody is determined by its light and heavy chain CDRs, the CDRs can be used to obtain antibody derivatives that retain antigen binding specificity. Examples of antibody derivatives include chimeric antibodies, humanized antibodies, and functional equivalents thereof. The following shows the light chain V region (SEQ ID NO: 25) and heavy chain V region (SEQ ID NO: 26) of the humanized 15A7 antibody, comprising SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6, respectively:

  The invention also features an isolated nucleic acid having a sequence that encodes one of the above immunoglobulin chains. The term “antibody” or “immunoglobulin chain” refers to an isolated polypeptide, ie, a polypeptide that is substantially separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide may constitute at least 50, 70, or 95% by dry weight of the purified preparation. The term “isolated nucleic acid” means a nucleic acid having a structure that does not match the structure of a naturally occurring nucleic acid or the structure of a naturally occurring fragment of a genomic nucleic acid. Thus, for example, the language may (a) have a sequence of a portion of a naturally occurring genomic DNA molecule, but not adjacent to both of the coding sequences adjacent to that portion of the naturally occurring genome molecule. DNA; (b) nucleic acid integrated into the vector or into prokaryotic or eukaryotic genomic DNA such that the resulting molecule does not match the naturally occurring vector or genomic DNA; (c) cDNA, genomic fragment, polymerase Another molecule such as a fragment produced by chain reaction (PCR), or a restriction fragment; and (d) a hybrid gene, ie a recombinant nucleotide sequence that is part of a gene encoding a fusion protein. The nucleic acids of the invention can be used to express the polypeptides of the invention. For the above purpose, an expression vector may be obtained by operably linking a nucleic acid to an appropriate regulatory sequence.

  By vector is meant a nucleic acid molecule that can transport other previously linked nucleic acids and that can self-replicate or integrate into host DNA. Examples include plasmids, cosmids, and viral vectors. The vectors of the present invention comprise a form of nucleic acid suitable for expressing the nucleic acid in a host. Preferably, the vector is operably linked to a nucleic acid sequence to be expressed by one or more regulatory sequences. Examples of regulatory sequences include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Regulatory sequences also include those that allow constitutive expression of nucleotide sequences in addition to tissue-specific regulatory and / or derived sequences. Such an expression vector is designed based on the selection of a host cell to be transformed and a desired expression level. An expression vector may be introduced into a host cell to produce the polypeptide of the present invention. The present invention also includes a host cell containing the nucleic acid. By host cell is meant a cell that contains exogenous coding or non-coding sequences. Exogenous sequences can be introduced into cells by calcium phosphate transfection, DEAE-dextran transfection, or electroporation. Suitable host cells include bacterial cells (eg, E. coli, Bacillus subtilis, and Salmonella typhimurium), yeast cells (eg, Saccharomyces cerevisiae and Saccharomyces cerevisiae cerevisiae cerevisiae). Schizosaccharomyces pombe), plant cells (eg, Nicotiana tabacum and Gossypium hirsutum), and mammalian cells (eg, mouse hybridoma cells, CHO cells) and 3T3 cells Can be mentioned.

  In order to produce the immunoglobulin chain of the present invention, host cells can be cultured under conditions capable of expressing the polypeptide encoded by the nucleic acid, and the polypeptide can be isolated from the culture. Alternatively, the nucleic acids of the invention may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  Antibodies are encompassed within the scope of the present invention. The antibody is formed by a first immunoglobulin chain and a second immunoglobulin chain, which contain the light and heavy chain CDRs of the mouse 15A7, 43B6, or 9F9 antibody, respectively. Preferably, the antibody is formed by the light and heavy chains of 15A7.

  (I) specifically binds to P-selectin glycoprotein ligand 1 without interfering with the binding between P-selectin glycoprotein ligand 1 and P-selectin, and (ii) P-selectin glycoprotein ligand of activated T cells Other antibodies that, when bound to 1, induce T cell death are also within the scope of the present invention. In one embodiment, the antibody specifically binds to human P-selectin glycoprotein ligand 1.

  Other antibodies that specifically bind to amino acids 115-126 of mature human P-selectin glycoprotein ligand 1 are further included within the scope of the present invention. Preferably, the antibody specifically binds to amino acid residues 117-123. More preferably, the antibody specifically binds to amino acid residues 119 to 121, which is a common sequence among all test epitopes. In practice, mutations in one or more of these three amino acid residues will result in a loss of antibody binding. As one example, this antibody induces death of activated T cells when bound to P-selectin glycoprotein ligand 1 of activated T cells.

  In one embodiment, one of the two antibodies described above comprises a light chain comprising SEQ ID NO: 1-3 and SEQ ID NO: 4-6 (eg, SEQ ID NO: 19 and 20, or SEQ ID NO: 25 and 26), respectively. Formed by chains and heavy chains.

  According to a further aspect, the invention features a method of inducing death of activated T cells. The method includes contacting one of the above three antibodies with activated T cells, wherein the antibody binds to activated T cells, thereby inducing cell death.

  The invention also features a method of modulating an immune response mediated by a patient's T cells. The method identifies (1) a patient having or at risk of having an immune response mediated by an excess of T cells, and (2) giving the patient one of the three antibodies. Having to administer an effective amount. By “excess T cell-mediated immune response” is meant a response caused by an excessive level of activated T cells. Excessive level means (1) a level that is higher than the normal level and (2) a level that is lower than the normal level but higher than the level desired by the individual. Examples of symptoms include inflammatory diseases, autoimmune diseases, allergic diseases, or T-cell cancer, in addition to situations where a patient has received or is scheduled to receive an allogeneic or xenograft.

  The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the detailed description.

DETAILED DESCRIPTION The present invention is capable of inducing apoptosis of activated T cells and depleting activated T cells, at least in part, by conjugating an antibody or derivative thereof to PSGL-1 of activated T cells. It is based on the unexpected discovery that it can. The antibodies and derivatives thereof are useful for treating conditions associated with excess or unwanted T cell mediated immune responses or T cell proliferation.

  Accordingly, the present invention features polypeptides comprising immunoglobulin light chain or heavy chain CDRs of anti-PSGL-1 antibodies, as well as nucleic acids that encode such polypeptides. Both immunoglobulin chains and nucleic acids can be used to make the antibodies and derivatives described above.

  The immunoglobulin chains of the present invention may be obtained as a synthetic polypeptide or a recombinant polypeptide. In order to prepare a recombinant polypeptide, the nucleic acid encoding it can be fused to a fusion partner such as glutathione-S-transferase (GST), 6x-His epitope tag (6x-His epitope tag), M13 gene 3 protein. (M13 Gene 3 protein), or other nucleic acid encoding immunoglobulin heavy chain constant region. The resulting fusion nucleic acid can be introduced into cells for the purpose of protein expression. The fusion protein can be isolated from the host cell by methods known in the art. The isolated fusion protein may be further processed, for example, by enzymatic digestion to remove the fusion partner and yield a useful recombinant polypeptide. Alternatively, the immunoglobulin chain may be obtained from a suitable host by activating endogenous expression of the nucleic acid encoding the chain.

  The amino acid composition of the immunoglobulin chain of the present invention may vary as long as it does not destroy the ability to form antibodies that can bind to PSGL-1. For example, such variants may contain one or more constitutive amino acid substitutions. “Constitutive amino acid substitution” is an amino acid residue substituted with an amino acid residue having a similar side chain. Amino acid residue groups having similar side chains are defined in the art. These groups include amino acids having base side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged side chains (eg, glycine, asparagine). , Glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (eg, Threonine, valine, isoleucine) and amino acids having aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain group. Alternatively, mutations may be introduced randomly along all or part of the polypeptide of the present invention, such as by saturation mutagenesis, and the resulting variants are similar to those described in the Examples below. Thus, the mutant of the present invention may be identified by screening for the ability to form an antibody capable of binding to PSGL-1. Thus, by way of example, the term “immunoglobulin chain comprising SEQ ID NO: 19” includes an immunoglobulin chain comprising a variant of SEQ ID NO: 19.

The above immunoglobulin chains and variants can be used to make the antibodies or derivatives thereof of the present invention. “Antibodies” are complete molecules such as Fab, F (ab ′) ₂ , Fv, scFv (single chain antibody), and dAb (domain antibody, Ward, et. Al. (1989) Nature, 341, 544). And even fragments thereof. An antibody derivative means a protein or protein complex of a polypeptide variant of the invention. The antibodies or derivatives of the invention can be made by expressing together polypeptides comprising the corresponding light and heavy chain CDRs in a suitable host cell as described in the Examples below. Alternatively, they can be made by known methods in the field of making monoclonal and polyclonal antibodies and fragments. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, New York.

  In order to produce the antibody of the present invention, PSGL-1 or an antigenic fragment thereof may be coupled to a carrier protein such as KLH, mixed with an adjuvant, and injected into a host animal. The antibody produced in the animal can then be purified by peptide affinity chromatography. Commonly used host animals include rabbits, mice, guinea pigs, and rats. The various adjuvants that can be used to increase the immunological response vary depending on the host species, Freund's adjuvant (complete and incomplete), inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions , Peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Human adjuvants that can be used include BCG (Bacilt Calmette-Guerin) and Corynebacterium parvum.

  Polyclonal antibodies, a heterogeneous population of antibody molecules, are present in the serum of immunized patients. Monoclonal antibodies, which are homogeneous populations of antibodies against a particular antigen, can be prepared using hybridoma technology. See, for example, Kohler et al. (1975) Nature 256, 495; Kohler et al. (1976) Eur. J. et al. Immunol. 6,511; Kohler et al. (1976) Eur. J. et al. Immunol. 6,292; and Hammerling et al. (1981) Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N .; Y. See In particular, monoclonal antibodies are techniques that provide for the production of antibody molecules by continuous cell systems in culture as described in US Pat. No. 4,376,110; human B cell hybridoma technology (Kosbor et al. (1983)). Colo et al. (1983) Proc. Natl. Acad. Sci. USA 80, 2026) and EBV hybridoma technology (Cole et al. (1983) Monoclonal Antibodies and RanT. , Pp. 77-96). Such an antibody may be an immunoglobulin class such as IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibody of the present invention may be cultured in vitro or in vivo. The ability to produce high titer monoclonal antibodies in vivo provides a particularly useful production method.

In addition, techniques developed for the production of “chimeric antibodies” can be used. For example, Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81,6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314,452. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (US Pat. Nos. 4,946,778 and 4,704,692) may be applied to produce phage libraries of single chain Fv antibodies. . Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge. In addition, antibody fragments can be obtained by known techniques. For example, such fragments include, but are not limited to, F (ab ′) ₂ fragments that can be produced by pepsin digestion of antibody molecules, and disulfide bridges of F (ab ′) ₂ fragments. There are Fab fragments obtained by The antibodies may also be humanized as described in the examples below or by known methods. For example, monoclonal antibodies having the desired binding specificity can be commercially humanized (Scotgene, Scotland; and Oxford Molecular, Palo Alto, Calif.). Fully humanized antibodies, such as those expressed in transformed animals, are encompassed within the scope of the present invention (see, eg, Green et al. (1994) Nature Genetics 7,13; and US Pat. No. 5,545,806). No. and 5,569,825).

  For example, death of activated T cells by contacting activated T cells in vitro with an antibody of the invention and administering an effective amount of an antibody to a patient in need of inducing death of activated T cells. A method for deriving is also within the scope of the present invention. A patient to be treated can be one that has been identified as having a risk associated with or at risk of having an immune response mediated by excess or unwanted T cells, such as an autoimmune disease, There may be patients suffering from transplant rejection, allergic disease, or T cell-induced cancer. The method can be performed alone or in combination with other drugs or therapies.

  The term “treatment” refers to a patient whose purpose is to cure, alleviate, reduce, repair, prevent or ameliorate a disease, symptoms of the disease, a medical condition secondary to the disease, or a predisposition to illness Administration of the composition. An “effective amount” is the amount of the composition that can produce a medically desirable result for the patient being treated.

  Examples of diseases to be treated include diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoporosis, and psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, self Immune thyroiditis, dermatitis (including atopic dermatitis and eczema-like dermatitis), psoriasis, Sjogren's syndrome, Crohn's disease, after ulcer, iritis, conjunctivitis, keratoconjunctivitis, type 1 diabetes, inflammatory bowel disease, Ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruption, leprosy reversal reactions, erythema nodosum erythema, autoimmune uveitis, allergic Encephalomyelitis, acute necrotizing hemorrhagic encephalomyelitis, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, erythroblastoma, idiopathic platelets Reduction, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, posterior uvea Transplant cases such as inflammation, interstitial pulmonary fibrosis, graft-versus-host disease, bone marrow transplantation, liver transplantation, or transplantation of any organ or tissue, including transplants using allogeneic or xenogeneic tissues, atopic Allergies such as allergies, AIDS, and T cell tumors such as leukemia or lymphoma.

  In one in vivo approach, a therapeutic composition (eg, a composition comprising an antibody of the invention) is administered to a patient. Usually, the antibody is suspended in a pharmaceutically acceptable carrier (eg, saline) and administered orally or by intravenous injection, or subcutaneously, intramuscularly, intrathecally, intraperitoneally, intrarectally. In addition, it may be injected or implanted into the vagina, into the nasal cavity, into the stomach, into the trachea, or into the lungs.

  The required dosage will depend on the choice of route of administration; the nature of the formulation; the nature of the patient's condition; the size, weight, surface area, age and sex of the patient; other drugs administered; and the judgment of the attending physician. A suitable dose is in the range of 0.01 to 100.0 mg / kg. Variations in the required dosage can be expected in view of the different available compositions and the different effectiveness of the various routes of administration. For example, oral administration is expected to require a higher dose than administration by intravenous injection. These dosage level changes can be adjusted using standard empirical procedures for optimization as is well understood in the art. Encapsulation of the composition in a suitable delivery vehicle (eg, polymeric microparticles or implantable device) may enhance the effectiveness of delivery, particularly oral delivery.

  Also included within the scope of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of an antibody of the invention. The pharmaceutical composition can be used to treat diseases as described above. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents.

  The pharmaceutical compositions of the present invention can be formulated in dosage forms for various routes of administration using known methods. For example, it may be formulated into capsules, gel seals, or tablets for the purpose of oral administration. Capsules may contain standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated according to known methods by compression mixing the composition with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The composition may also be administered in the form of a capsule containing a hard shell tablet or binder, such as lactose or mannitol, a known filler, and a tableting agent. The pharmaceutical composition may be administered by a parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic solutions or active agents 5% glucose, or other known pharmaceutically acceptable excipients. Cyclodextrins, or other solubilizers known to those skilled in the art, may be used as pharmaceutical excipients for the purpose of delivering therapeutic agents.

  The effectiveness of the composition of the present invention can be evaluated both in vitro and in vivo. For example, see the examples below. Briefly, the composition may be tested for the ability to induce death of activated T cells in vitro. For in vivo studies, the composition is injected into an animal (eg, a mouse model) and its therapeutic effect is evaluated. Based on the results, an appropriate dosage range and administration route may be determined.

  The following specific examples are to be construed as merely illustrative in detail and are not intended to limit the remainder of the disclosure in any way. Without further explanation, it is considered that those skilled in the art can fully utilize the present invention based on the description of the present specification. All publications listed herein are hereby incorporated by reference in their entirety.

Example 1: Mouse monoclonal antibodies 15A7, 43B6, and 9F
Generation of anti-PSGL-1 antibody A mouse monoclonal antibody that specifically binds to human PSGL-1 (hCD162) was obtained using standard techniques. More specifically, mice were immunized with a membrane fraction of PHA-activated human T cells and necropsied to obtain a hybridoma cell line. The resulting hybridoma cell line supernatant was screened for binding to CHO cells stably expressing hCD162. Cell lines producing antibodies that bind to CHO cells expressing hCD162 but not to parental CHO cells were identified, subcloned and further analyzed as described below.

  m152-15A7, m166-43B6, and m128-9F9 were included in the identified system. These produced IgG1 antibodies 15A7, 43B6, and 9F9, respectively. Immunoblotting assays show that these three antibodies destroyed from lysates of activated T cells produce proteins that can be detected by anti-hCD162 antibodies (kpl-1, PharMingen, San Diego, Calif.). It was done.

  The above three antibodies were tested for the ability of activated T cells to induce apoptosis. Culture supernatants containing monoclonal antibodies secreted by the three hybridoma cell lines were each treated for 6 hours with inactivated human T cells (day 0) or in vitro activated human T cells (day 7). Incubated with. The cells were then stained with annexin V and subjected to FACS analysis. Cells positive for CD3 were gated and either the number of human T cells activated in vitro or the number of resting human T cells was counted. Apoptotic cells were positive for annexin V staining. Table 1 summarizes the percentage of apoptotic T cells occupied in the total scanned T cells.

  From these results, the mouse 15A7, 43B6, and 9F9 antibodies are (1) specific for hCD162, (2) bind to human activated T cells and do not induce apoptosis of resting human T cells, It has been shown that apoptosis of activated T cells induces.

  In addition, apoptosis assays were performed with PHA-activated human peripheral blood mononuclear cells (PMBC). The antibody was found to induce apoptosis of activated T cells only and not apoptosis of resting T cells, B cells, or neutrophils.

  It is known that T cell depleting antibodies, such as anti-CD3, can induce the production of soluble factors. Treatment with such antibodies generally causes harmful cytokine syndrome. To test whether anti-PSGL-1 antibody causes cytokine-related side effects, freshly isolated human PBMC were cultured with 15A7 for 24, 48, or 72 hours. Next, cytokine levels in the supernatant were measured. Significant amounts of IL-2, TNF-a, and IFN-a were produced in PHA-activated PBMC (positive control), but the levels of the cytokines from cells treated with 15A7 were undetectable . These results suggest that anti-PSGL-1 has no or little effect on resting peripheral blood cells in both apoptosis induction and cell activation.

  Since the antibody selectively induces apoptosis of activated T cells without adversely affecting resting T cells or other immune cells, anti-CD3 or immunosuppression can be achieved by administering such antibodies to patients. It is thought not to cause lymphopenia like drugs or widespread immunodeficiency.

Epitope mapping of anti-CD162 antibody To perform mapping of the binding epitopes of mouse 15A7, 43B6, and 9179 with human CD162, a series of fusion proteins encompassing various regions of human CD162 were expressed and purified. The interaction of the fusion protein with these monoclonal antibodies was tested by sandwich enzyme immunosorbent assay (ELISA).

  Briefly, fragments encompassing various regions of the human CD162 gene were expressed in E. coli as fusion proteins with human immunoglobulin gamma 1 heavy chain constant regions. CDNA encoding the human immunoglobulin gamma 1 heavy chain constant region was amplified by PCR using primers with a BglII site and a BamHI site. The PCR product was cleaved with BglII and BamHI and further subcloned into the pET-32a vector (Novagen) previously digested with the same enzymes. Next, cDNAs encoding various regions of hCD162 were amplified by PCR using primers having an NdeI site at the 5 'end and a BglII site at the 3' end. The PCR product was cleaved with the corresponding enzyme and fused in frame to the sequence encoding the human immunoglobulin gamma 1 heavy chain constant region of the pET-32a vector. The primers used in each construction are listed in Table 2, and the primer sequences are listed in Table 3.

The expression construct was transformed into E. coli strain BL21 (DE3). After IPTG (2 mM) induction for 6 hours, the transformed cells were collected and resuspended in PBS. Cells were sonicated and centrifuged at 14,000 g for 10 minutes, and the resulting supernatant was collected for fusion protein purification. More specifically, the supernatant was first incubated with protein G or protein A beads for 3 hours at 4 ° C. Then, centrifuged the beads in 3,000 g, washing buffer I (0.05% Triton X-100,50mM Tris-HCl, pH 8.5,400mM NaCl, 1mM CaC1 2 and 1 mg / ml OVA) and washing Each was washed 5 times with buffer II (0.05% Triton X-100, 50 mM Tris-HCl, pH 8.5 and 150 mM NaCl). Further, the bound protein was eluted with an elution buffer containing 0.1 M glycine-HCl, pH 2.7 and neutralized with 1 M Tris-HCl, pH 8.6. Total purified fusion protein was quantified by Bio-Rad protein assay (Bio-Rad Laboratories, Cat. No. 500-0006) and confirmed by SDS-PAGE.

In order to study the interaction of the hCD162 fragment with each of 15A7, 9F9, and 43B6, a sandwich ELISA was performed. A 96 well microtiter plate was coated with goat anti-human IgG (Southern Biotechnology, Cat. No. 2040-01) antibody (2 μg / ml, 50 μl / well) at 4 ° C. overnight. Plates were blocked by incubating with 0.25% BSA in PBS (150 μl / well) for 1 hour at 37 ° C. The blocked plates were then incubated with fusion proteins containing various fragments of human CD162 (2 μg / ml) for 2 hours at room temperature. After 4 washes with PBS containing 0.05% Tween 20 (PBST), the plates were incubated with test antibody (2 μg / ml) for 1.5 hours at room temperature. After incubation, the plate was washed 4 times with PBST. Further, goat anti-mouse IgG (Southern Biotechnology, Cat. No. 1031-04) conjugated with alkaline phosphatase diluted 1/3000 times 50 μl was added to each well and the plate was incubated at 37 ° C. for 1 hour. 50 μl alkaline phosphatase substrate solution (one alkaline phosphatase substrate tablet dissolved in 5 ml substrate buffer containing 0.012 M Na ₂ CO ₃ , 0.16 M NaHCO ₃ and 1 mM MgCl ₂ at pH 8.6) And the enzyme reaction was carried out, and the absorbance at 405 nm was measured.

  Since it was found that 43B6 and 9F9 can interact with all fusion proteins containing residues 50-60 of mature human CD162, the epitope of 43B6 and 9F9 is between residues 50-60 It was suggested to be located. Unlike 9F9 and 43B6, only 15A7 was not a fusion protein covering residues 42-119, but rather a fusion protein covering residues 42-319, so the epitope of 15A7 is 119- It was suggested to be located between the 319th residue. Next, the position of the 15A7 epitope narrowed to between positions 115-126. Since the interaction between 15A7 and the fusion protein disappeared by changing one amino acid at position 120 (Glu → Arg), the primary contact domain of 15A7 of human CD162 is located at or near position 120. Residue Glu was suggested to be essential for the interaction.

  Fusion proteins covering various human CD162 regions were also expressed in mammalian cells and tested for interaction with 15A7. Fragments covering these regions were expressed in mammalian cells as fusion proteins with human immunoglobulin gamma 1 heavy chain constant regions. First, cDNA encoding human immunoglobulin gamma 1 heavy chain constant region was inserted into pcDNA3 vector (Invitrogen). Second, cDNAs encoding various regions of hCD162 were amplified by PCR using primers that introduced a BamHI site at the 5 'end and an XhoI site at the 3' end. These PCR products were cleaved with the corresponding enzymes and subcloned into pcDNA3 vector containing human immunoglobulin gamma 1 heavy chain constant region. The names and sequences of each primer are listed in Tables 2 and 3 above.

  The mammalian expression vectors described above were transiently transfected into COS-7 cells with Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) according to the manufacturer's instructions. Transfected cells were grown in ultra-low Ig medium (Invitrogen, Cat. No. 16250-078). The expressed protein was purified and subjected to a sandwich ELISA as described above.

  ELISA results show that only fusion proteins containing residues 94-148 were able to interact with 15A7. These results are consistent with the notion that 15A7 is located between residues 115-126.

  All of the above results indicate that the epitopes of 9F9, 43B6, and 15A7 are protein dependent rather than carbohydrate modification dependent because all three antibodies bind to the bacterially expressed fusion protein. 15A7, 9F9, and 43B6 also show similar properties in terms of binding specificity and fusion of activated T cell apoptosis, suggesting that they function in different domains of human CD162 and behave differently.

Example 2: Chimeric antibodies 15A7, 43B6, and 9F9
Cloning of light chain and heavy chain variable regions of anti-CD162 antibodies cDNAs encoding the light chain and heavy chain variable regions (V _L and V _H ) of antibodies 15A7, 43B6, and 9F9 were anchored PCR methods. Amplified by The 3 ′ primer hybridized with the C region and the 5 ′ primer hybridized with the G tail bound to the cDNA using terminal deoxytransferase. The PCR fragment was cloned into the pCRII vector (Invitrogen). Several independent clones were sequenced and compared for each chain. Sequences represented by most independent clones were selected. Next, the translated amino acid sequence was analyzed, and it was confirmed that the predetermined sequence had specific mouse light chain or heavy chain V region characteristics and belonged to a specific subtype. In addition, complementarity determining regions (CDRs) were identified by comparing the translated amino acid sequence with the consensus sequence of each subtype. The names and sequences of each primer used are listed in Tables 2 and 3 above. The deduced amino acid sequences (SEQ ID NOs: 19-24) of the light and heavy chain V regions of 15A7, 43B6, and 9F9 are summarized and shown.

Chimeric Antibody To obtain a vector for expressing the chimeric antibody, a cDNA encoding the _VL and _VH regions of 15A7, 43B6, and 9F9 is included so that it contains a 5 ′ signal peptide sequence and a 3 ′ splice donor signal. Amplified by PCR using primers. The primers also had XbaI sites introduced at both ends of the PCR product, which were further cleaved with the XbaI enzyme and ligated into a pVk, pVg1, pVg2, or pVg4 vector digested with XbaI. More specifically, the 15A7, 43B6, and 9F9 _VL region cDNAs were subcloned into plasmid pVk. This plasmid contained the CMV plasmid and sequences encoding the human light chain constant region. The 15A7, 43B6, and 9F9 VH region cDNAs were subcloned into plasmids pVg1, pVg2, or pVg4. Each of the three plasmids had a CMV promoter. They also contained human heavy chain constant regions of IgG1, IgG2, and IgG4, respectively.

  Each of the plasmids encoding the light chain was transfected into COS-7 cells together with the plasmid encoding the heavy chain. The supernatant of the transfected cells was collected. The chimeric antibody in the supernatant was analyzed for its ability to bind to human CD163 and to induce apoptosis of activated T cells.

  All of the chimeric antibodies made from 15A7, 43B6, and 9F9 were found to bind to Sp2 / 0 transfectants that stably express human CD162, but not to the parent Sp2 / 0 cells. It was suggested that these retain the human 162 binding ability specificity. Furthermore, the chimeric antibodies were found to induce apoptosis of T cells activated for 7 days, suggesting that they retain the function of the mouse counterpart as well.

Humanized antibodies Humanized antibodies were generated using mouse 15A7 by grafting CDRs onto the human framework. In order to preserve binding affinity and specificity, it is important to preserve the structure of the V region when grafting CDRs onto the human framework. In order to select an appropriate framework donor, the amino acid sequences of the mouse 15A7 light chain and heavy chain V regions were compared to the amino acid sequences of 50 mouse antibodies that were previously humanized.

  The mouse antibody, mDREG-55, was found to have high sequence homology with the V region of mouse 15A7 in both the light and heavy chains. The sequence alignment of mouse 15A7 to this mDREG-55 antibody is listed below (CDR is highlighted).

Mouse DREG-55 is an IgG1 monoclonal antibody against L-selectin. The sequences of the _VL and _VH regions of mouse 15A7 are 64.3% (framework only: 73.8%) and 70% (framework only: 81.6%) relative to that of mouse DREG55, respectively. Homology. Humanized DREG-55 (HuDREG-55) has already been constructed from the human antibody Gal using framework sequences of the _VL and _VH regions. Therefore, to humanize mouse 15A7, the human Gal light and heavy chain framework sequences were used to replace the mouse 15A7 counterpart.

  The humanized 15A7 light chain and heavy chain variable regions were each assembled by 4 pairs of synthetic oligonucleotides (approximately 80 bases long). Each pair of oligonucleotides overlapped by about 20 nucleotides. Nucleotide sequences were selected and synthesized to coat the protein sequence of the humanized variable region containing the signal peptide. Gene assembly and amplification was performed in four steps: (1) annealing 4 pairs of complementary oligonucleotides and extending with Klenow fragment in 4 separate reactions; (2) resulting 4 Two dsDNA fragments are mixed, denatured, reannealed and extended in two separate reactions; (3) the resulting two dsDNA fragments are mixed, denatured, Annealed and further extended to produce the final full-length dsDNA; further (4) the resulting DNA was amplified by PCR using primers to introduce XbaI sites at both ends. The PCR fragment was then cut with XbaI and inserted into each of the pVk and pVg4 vectors digested with XbaI. In addition, the Gal residue was changed to that of mouse 15A7 (ie, I62V and D74H) at positions where the interaction between the CDR and the framework was considered important. The alignment of mouse 15A7 and humanized 15A7 (Hul5A7) to mDREG-55 is shown below, with V62 and H74 underlined.

  The plasmid thus obtained encoded the heavy and light chains of humanized 15A7. These plasmids were then transfected together into COS-7 cells. Furthermore, the exhausted supernatant from the cultured cells was collected. Humanized 15A7 in the supernatant was tested for its ability to bind to CHO transfectants that stably express hCD162 and to induce apoptosis of T cells activated for 7 days. The results show that this retains these capabilities.

Preparation of chimeric and humanized antibodies Cells producing humanized and chimeric antibodies were obtained. More specifically, Sp2 / 0 cells (Sp2 / 0-Agl4; ATCC CRL 1581) were used with a Gene Pulser apparatus (Bio-Rad Laboratories) with a capacitance of 360 V and 25 μF according to the manufacturer's instructions. The appropriate plasmid was stably transfected by electroporation. Prior to transfection, the plasmid was linearized by digestion with BamHI enzyme. All transfections were performed with 10 ⁷ cells and 20 μg of each plasmid DNA in PBS. Cells obtained from each transfection were seeded in 96-well tissue culture plates. After 48 hours, selective medium (DMEM 10% FBS / hypoxanthine / thymidine medium supplement) and 1 μg / ml mycophenolic acid were applied. Antibody producing cells were screened and isolated by examining the presence of the antibody in the culture supernatant by ELISA.

Isolated cells were cultured in serum-free or low Ig medium and the culture supernatant was collected. The antibody was purified by passing through a column of staphylococcal protein A-Sepharose CL-4B. Wash Buffer I (0.05% TritonX-100,50mM Tris -HCl, pH 8.5,400mM NaCl, 1mM CaC1 2 and 1 mg / ml OVA) and washing buffer II (0.05% TritonX-100,50mM Tris- After washing 5 times each with HCl, pH 8.5 and 150 mM NaCl), the bound antibody is eluted with an elution buffer containing 0.1 M glycine-HCl, pH 2.7, and 1 M Tris-HCl, pH Neutralized at 8.6.

Affinity measurement The binding affinity of the mouse, chimeric, and humanized 15A7 antibodies was measured by competitive binding.

Mouse 15A7 was biotinylated with the EZ-Link Sulfo-NHS-Biotin system (Pierce Biotechnology, Cat. No. 21217) (EZ-Link Sulfo-NHS-Biotin system). Briefly, 0.5 mg (3.3 × 10 ⁻⁶ nmole) of mouse 15A7 was dissolved in 187 μl PBS and mixed with 6.8 × 10 ⁻⁵ nmole sulfo-NHS-biotin. The mixture was then incubated on ice for 2 hours before free biotin was removed by dialysis against PBS overnight at 4 ° C. The biotin-labeled mouse 15A7 thus obtained was stored at 4 ° C. until used.

Sp2 / 0 transfectants that stably express human CD162 were used as a human CD162 antigen source. Biotin-labeled mouse 15A7 was used as a tracer. Increase the amount of competitor antibody (mouse, chimeric or humanized 15A7), mix with 35 ng of biotinylated mouse 15A7 and express 1 × 10 ⁵ CD162 for 1.5 hours at 4 ° C. with constant shaking Incubated with Sp2 / 0 cells. After washing, a secondary antibody, Streptavidin-PE (Becton Dickinson Immunocytometry System Inc. Cat. No. 349023) was added to the mixture. After 45 minutes incubation at 4 ° C., the cells were washed again, resuspended in 300 μl PBS-1% FBS and subjected to FACS analysis.

The half-value competitive concentration of chimera and humanized 15A7 is approximately 5.71 μg / ml and 4.51 μg / ml, respectively, while the half-value competitive concentration of mouse 15A7 is 3.72 μg / ml. It turns out that. These results suggested that the affinity of mouse, chimera, and humanized 15A7 is comparable. In other words, the binding affinity (Ka) for chimeric and humanized 15A7 is 2.62 × 10 ⁷ M ⁻¹ and 3.33 × 10 ⁷ M ⁻¹ , respectively, whereas the binding affinity for mouse 15A7 The sex (Ka) is 4.03 × 10 ⁷ M ⁻¹ .

Competition analysis A competition analysis was performed to study the interaction among the three mouse antibodies, PSGL-1 and P-selectin.

  P-selectin is the major high-affinity ligand for PSGL-1 in most leukocytes. To examine whether the three antibodies interfere with the binding of P-selectin to PSGL-1, the binding of purified human P-selectin to activated T cells was determined in the presence of the above three antibodies. It was measured. KPL-1, which is known to block the interaction of P-selectin and PSGL-1, was used as a positive control.

  Human PBMC were activated with 1% PHA for 2 days and maintained in medium containing IL-2 for 3 days. Cells were incubated for 30 minutes with 9F9, 15A7, 43B6, KPL-1 (an antagonist of PSGL-1), or a control antibody (9E10) added dropwise, then recombinant human P-selectin (1.25 μg / ml) Was added. Binding of P-selectin to activated T cells was measured by anti-P-selectin-FITC analyzed by FRCS.

  Consistent with previous reports, KPL-1 almost completely inhibited P-selectin binding to activated T cells at low concentrations (0.31 μg / ml). 43B6 blocked P-selectin binding to activated T cells with the same efficacy as KPL-1, but a higher concentration of 9F9 was required to achieve the same effect. Indeed, 0.08 μg / ml KPL or 43B6 was required to inhibit 50% binding. In contrast, 5 μg / ml 9F9 was required. Furthermore, 15A7 showed no inhibitory effect on P-selectin binding even at 20 μg / ml. Surprisingly, this promoted the binding of P-selectin to PSGL-1. These results suggest that 15A7 and P-selectin bind to different motifs of PSGL-1 in activated T cells.

  Due to the fact that 15A7 does not compete with P-selectin for PSGL-1, in vivo administration of 15A7 is not expected to affect innate immunity by preventing leukocyte P-selectin-dependent recruitment Is suggested.

  PSGL-1 has been reported to be expressed at low levels in platelets. The effect of 15A7 antibody on platelets was examined. The antibody was found not to promote or inhibit aggregation of human platelets.

Example 3: Hamster monoclonal antibody TAB4 against mouse PSGL-1
TAB4, a monoclonal antibody against mouse PSGL-1, was prepared in the same manner as described in Example 1. This induced apoptosis of T cells in vitro and depleted T cells in vivo. In order to determine whether or not the binding between mouse PSGL-1 and mouse P-selectin is prevented, competition analysis was performed in the same manner as described in Example 2. TAB4 was found not to inhibit the binding of mouse P-selectin to mouse PSGL-1 even at a high concentration of 20 μg / ml.

Example 4: Mouse monoclonal antibodies 4B7, 5C4, 12E7, 14B3, 17E5, and 18D12
Further characterization of monoclonal antibodies 4B7, 5C4, 12E7, 14B3, 17E5, and 18D12 against human PSGL-1. When bound to activated T cells, they all induced death of activated T cells. In order to determine whether to block the interaction between PSGL-1 and P-selectin, competition analysis was performed in the same manner as described in Example 2. These antibodies were found to show little, if any, inhibitory effect on human P-selectin binding to human PSGL-1 even at the highest test concentration (5 μg / ml).

Other Embodiments All aspects disclosed herein may be combined in any combination. Each aspect disclosed herein may be replaced with another aspect serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each aspect disclosed is one example of a generic group of equivalent or similar aspects.

  From the above description, those skilled in the art can easily confirm the essential features of the present invention, and various changes and modifications of the invention can be made without departing from the concept and scope of the present invention. Can be applied to conditions. Thus, other embodiments are also within the scope of the present invention.

Claims (40)

  An immunoglobulin chain having SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, or SEQ ID NO: 16-18, respectively.   The immunoglobulin chain of claim 1, wherein the immunoglobulin chain has SEQ ID NO: 1-3 or SEQ ID NO: 4-6.   The immunoglobulin chain of claim 1, wherein the immunoglobulin chain has SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26.   4. The immunoglobulin chain of claim 3, wherein the immunoglobulin chain has SEQ ID NO: 25 or 26.   The first immunoglobulin chain of claim 1 and the second immunoglobulin chain of claim 1, wherein the first chain is a light chain, and the second chain is a heavy chain. The chains comprise SEQ ID NO: 1-3 and SEQ ID NO: 4-6; SEQ ID NO: 7-9 and SEQ ID NO: 10-12; or SEQ ID NO: 13-15 and SEQ ID NO: 16-18, respectively. antibody.   6. The antibody of claim 5, wherein the light and heavy chains comprise SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6, respectively.   6. The antibody of claim 5, wherein the light and heavy chains comprise SEQ ID NOs: 19 and 20, 21 and 22, 23 and 24, or 25 and 26, respectively.   8. The antibody of claim 7, wherein the light and heavy chains comprise SEQ ID NOs: 25 and 26, respectively.   Binding of activated T cells to P-selectin glycoprotein ligand 1 induces death of activated T cells, without interfering with the binding of P-selectin glycoprotein ligand 1 and P-selectin P-selectin glycoprotein ligand An antibody that specifically binds to 1.   10. The antibody of claim 9, wherein the antibody binds to human P-selectin glycoprotein ligand 1.   10. The antibody of claim 9, wherein the antibody comprises a light chain and a heavy chain comprising SEQ ID NO: 1-3 and SEQ ID NO: 4-6, respectively.   11. The antibody of claim 10, wherein the light and heavy chains comprise SEQ ID NOs: 25 and 26, respectively.   An antibody that specifically binds to amino acid residues 115 to 126 of human P-selectin glycoprotein ligand 1.   The antibody according to claim 13, wherein the antibody specifically binds to amino acid residues 117 to 123.   The antibody according to claim 14, wherein the antibody specifically binds to amino acid residues 119 to 121.   14. The antibody of claim 13, wherein the antibody comprises a light chain and a heavy chain comprising SEQ ID NO: 1-3 and SEQ ID NO: 4-6, respectively.   14. The antibody of claim 13, wherein the antibody induces death of activated T cells when bound to P-selectin glycoprotein ligand 1 of activated T cells.   18. The antibody of claim 17, wherein the light and heavy chains comprise SEQ ID NOs: 1-3 and SEQ ID NOs: 4-6, respectively.   9. Contact of an activated T cell with the antibody of claim 8, wherein the binding of the antibody to the activated T cell induces the death of the activated T cell. Guidance method.   An antibody according to claim 9 in contact with activated T cells, wherein the binding of said antibodies to activated T cells induces death of activated T cells; Guidance method.   14. The method according to claim 13, comprising contacting the antibody of claim 13 with an activated T cell, wherein the binding of the antibody to the activated T cell induces the death of the activated T cell. Guidance method. Identifying a patient having or at risk of having an immune response mediated by excess T cells;
Administering an effective amount of the antibody of claim 8 to the patient,
Methods of modulating immune responses mediated by patient T cells.   23. The method of claim 22, wherein the symptom is an inflammatory disease, an autoimmune disease, an allergic disease, or a T cell cancer.   24. The method of claim 23, wherein the patient has received or is scheduled to receive an allogeneic or xenograft. Identifying a patient having or at risk of having an immune response mediated by excess T cells;
Administering an effective amount of the antibody of claim 9 to the patient,
Methods of modulating immune responses mediated by patient T cells.   26. The method of claim 25, wherein the symptom is an inflammatory disease, an autoimmune disease, an allergic disease, or a T cell cancer.   27. The method of claim 26, wherein the patient has received or is scheduled to receive an allogeneic or xenograft. Identifying a patient having or at risk of having an immune response mediated by excess T cells;
Administering an effective amount of the antibody of claim 13 to the patient,
Methods of modulating immune responses mediated by patient T cells.   29. The method of claim 28, wherein the symptom is an inflammatory disease, an autoimmune disease, an allergic disease, or a T cell cancer.   30. The method of claim 29, wherein the patient has received or is scheduled to receive an allogeneic or xenograft.   An isolated nucleic acid having a sequence encoding the immunoglobulin chain of claim 1.   An isolated nucleic acid having a sequence encoding the immunoglobulin chain of claim 3.   32. A vector comprising the nucleic acid of claim 31.   A vector comprising the nucleic acid according to claim 32.   A host having a nucleic acid encoding SEQ ID NO: 1-3, SEQ ID NO: 4-6, SEQ ID NO: 7-9, SEQ ID NO: 10-12, SEQ ID NO: 13-15, or SEQ ID NO: 16-18 cell.   36. The host cell according to claim 35, wherein the cell is a bacterial cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell.   37. The host cell according to claim 36, wherein the mammalian cell is a hybridoma cell.   A host cell having a nucleic acid encoding SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26.   40. The host cell of claim 38, wherein the cell is a bacterial cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell.   40. The host cell of claim 39, wherein the mammalian cell is a hybridoma cell.